1. Field of the Invention
The present invention relates generally to the fields of molecular biology and genetics. More specifically, the present invention relates to a novel DNA sequences encoding a mouse phosphotriesterase-related protein and novel uses thereof.
2. Description of the Related Art
Polycystic kidney disease (PKD) is characterized by the development of innumerable, large, fluid-filled cysts from the nephrons and collecting ducts of affected kidneys. Enlargement of cysts is thought to interfere with functioning of the normal renal parenchyma, which eventually leads to renal failure. In humans, PKD can be inherited as an autosomal dominant (ADPKD) or an autosomal recessive trait (ARPKD). ADPKD is a common disease, affecting 1 in 500-1,000 individuals, and contributes significantly to the number of patients on long-term dialysis. ARPKD is less common, occurring in 1 in every 6,000-14,000 live births, but is usually fatal in infancy. To date, the factors causing the initiation of renal cysts and their progressive enlargement, and the development of azotemia and renal failure are not known. Biochemical and histological studies have identified cbaracteristics common to all cysts in polycystic kidney disease: abnormal cell proliferation, basement membrane alterations, and changes in cell polarity and transport or secretion. Considerable work, has focused on the potential involvement, of these factors in cyst formation and progression toward renal failure. Genetic approaches also have been utilized to identify the defective gene(s) in this disease in hopes of determining the primary defect in PKD. Although a number of genes showing abnormal expression PKD in human and animal models have been identified. The relationship of these genes and the common characteristics of renal cysts is not apparent.
The C57BL/6J-cpk mouse has been utilized as a model for human ARPKD. PKD in this animal model is inherited as an autosomal recessive trait and is characterized by the rapid development primarily of collecting duct cysts resulting in renal failure and death of the affected mice by 3-4 weeks of age. The cpk gene has been mapped to mouse chromsome 12, but has not been isolated and identified. Previous work has demonstrated the abnormal expression of different genes in the polycystic kidneys of cpk mice. These genes include the proto-oncogenes, c-fos, c-myc, and c-K1-ras; preproEGF; histone H4; beta-actin; and the developmentally regulated gene, sulfated glycoprotein 2 (SGP-2).
Nephrotoxic injury causes kidney tubule damage and necrosis, followed by repair of the damage and regeneration of normal renal function. Folic acid in high concentrations causes renal injury and is one of the most potent known stimuli for cell proliferation in the rodent kidney. The administration of a single large dose of folic acid by intraperitoneal injection is followed within hours by evidence of decreasing kidney function and by a number of cellular and molecular events consistent with the onset of kidney regeneration. Following folic acid-induced acute renal injury, there is an increased rate of cell division. The early phase of the response to folic acid treatment is an ordered sequence of expression of cell-cycle specific genes. There is a rapid and transient increase in the level of c-fos mRNA which peaks at 3 hours following folic acid treatment, followed by increases in c-myc, c-Ki-ras, c-Ha-ras and beta-actin mRNAs. These events precede or coincide with an increase in histone H4 mRNA at 24-36 hours following folic acid treatment. The temporal pattern of induction of these mRNAs suggests that the kidney responds very rapidly to folic acid, followed by the synchronized progression of kidney cells through the Gl phase of the cell cycle and ultimately through cell division. Renal injury has been shown to cause changes in the expression of a number of genes, including early response and cell cycle-regulated genes (c-fos, JE, KC, egr-1, c-myc, c-Ki-ras, c-Ha-ras, beta-actin, histones H2b and H4), growth factors and their receptors (HGF, c-met, EGF), the enzyme superoxide dismutase, and secreted proteins (renin, endothelin, Tamm-horsfall/uromodulin, SGP-2/clusterin/TRPM-2). While some of these genes appear to be associated with the cell proliferation required for kidney repair, a decrease in EGF or, an increase in SGP-2 has no known role in the injury process or in the subsequent regeneration.
The bacterial phosphotriesterases from Flavobacterium and Pseudoraonas diminuta are enzymes encoded by naturally occurring plasmids found in these two organisms. The protein coding regions of the genes in these two bacterial species are identical at the nucleotide sequence and amino acid sequence levels. The protein is a zinc metalloenzyme that has a broad substrate specificity, catalyzing the hydrolysis of organophosphate-triester compounds. Among the substrates of the enzyme are the insecticides parathion and paraoxon, and they have received a great deal of attention for their possible industrial, agricultural, and medical applications as a possible detoxifying enzyme or enzyme that may confer protection to a toxic overdose of organophosphates. The enzyme goes by a variety of names inclucling parathion hydrolase, organophosphorous acid anhydrase, aryldialkylphosphatase and phosphotriesterase. There is no known eukaryotic protein that is structurally or evolutionarily related to the Flavobacterium/Pseudomonas enzyme.
Arylesterases that degrade paraoxon have been isolated from liver microsomes and cytosol and from blood plasma of human, rabbit and rat. These enzymes, called paraoxonases, are synthesized predominantly, if not exclusively, in the liver and have different properties from the bacterial parathion hydrolase (phosphotriesterase). Furthermore, the human and rabbit cDNAs for serum paraoxonase/arylesterase have been isolated and characterized and are unrelated to both the Flavobacterium or Pseudomonas phosphotriesterase.
The prior art is deficient in the lack of effective means of detecting the polycystic kidney disease and renal failure. The present invention fulfills this longstanding need and desire in the art.